The research proposed here is a natural extension of the biophysical studies our lab has carried out over many years on the folding and assembly of membrane proteins (MPs), ranging from fundamental interactions of peptides with lipid bilayers to measurements of the co-translational insertion of transmembrane (TM) helices into microsomal membranes by the Sec61 translocon in collaboration with the von Heijne lab. The latter studies yielded the first biological hydrophobicity scales. We are now engaged in studies of TM helix stability in the inner membranes living E. coli using single-span MPs (S-SMPs) to take advantage of the possibility of inserting TM helices along two different pathways: the SecA post-translational pathway or the co-translational signal recognition particle (SRP) pathway. Our global objective is to connect quantitatively the physicochemical principles of TM helix stability with the molecular code for transmembrane helix recognition by translocons. Solving this problem will lay a necessary foundation for predicting 3D structure from amino acid sequence, which will provide insights into membrane protein misfolding disorders, such as cystic fibrosis. The molecular code is described by the apparent free energy of transfer (?Gapp) of an ?-helix between translocon and membrane bilayer whereas physicochemical stability is generally described by the free energy of transfer (?Gwbi) between water and bilayer. ?Gapp determines whether a nascent helix enters the bilayer from the translocon; ?Gwbi determines whether it will stay there. We will pursue several approaches to establish a firmer connection between ?Gwbi and ?Gapp. One approach to connecting the free energies is purely biological. We have discovered, using chimeras of the S-SMP CadC with designed TM segments, that some TM segments drop out of the E. coli inner membrane into the cytoplasm when the periplasmic domain is cleaved by signal peptidase. That is, some TM segments are metastable and remain in the membrane only because the water-soluble periplasmic domain cannot cross the membrane. This remarkable phenomenon will allow us to connect ?Gapp to ?Gwbi directly in a living biological system by establishing a threshold for stability. Our work on the metastability of CadC TM segments has raised fundamental questions about the targeting of S-SMPs that demand investigation. We have determined that CadC requires SecA for insertion via SecYEG, but that insertion is modulated by SRP. This effect is part of a larger problem: For multi-span membrane proteins, SecA is required for the transport of periplasmic domains containing 30 or more amino acids. Just how SecA and SRP cooperate to achieve this is completely unknown. We hypothesize the answer lies in understanding to the SecA and SRP pathways. We have developed new methods that (1) guarantee insertion by SecA alone of S-SMPs and (2) allow us to examine the interplay between signal-sequence and TM-segment hydrophobicities during pathway selection. These ideas provide the rationale for our four specific aims: 1. Establish a post-insertion stability threshold for TM segments (?Gwbi) and an in vivo a biological hydrophobicity scale for SecA-driven TM insertion (?Gapp). 2. Determine the dependence of pathway selection-SRP or SecA-on the relative hydrophobicities of single TM segments and signal sequences using our two new methods. 3. Use molecular dynamics simulations in conjunction with molecular biology experiments to establish a connection between the dynamics and electrostatics of translocons that accompany changes in ?Gapp caused by massive mutations of the Sec61 pore ring that lies at the center of Sec61. 4. Develop physical and computational methods for obtaining direct physicochemical data on ?Gwbi using synthetic TM segments based upon TM segments used in determinations of ?Gapp.